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Genetic toxicity in vitro

Description of key information
Concerning the bacterial gene mutation endpoint, the key study Ames test was negative. This study was conducted according to current test guidelines and GLP with 99.7% pure IPHA. Two of the supporting Ames studies (1986: Klimisch 2 and 1985: Klimisch 4) were negative as well. One study performed with a 15% watery solution of IPHA (Klimisch 4) reported mutagenicity. For mammalian cell gene mutation, one HGPRT assay was reported negative (1985: Klimisch 4), another HGPRT assay indicated a positive response (Haas-Jobelius et al. 1991: Klimisch 2), and the most recent CHO&HGPRT assay (2014: Klimisch 1) was negative. An in vitro rat hepatocyte UDS DNA repair assay also conducted by Haas-Jobelius 1991 was negative.  The clastogenicity endpoint was addressed by an in vitro V79 micronucleus assay by Haas-Jobelius et al. (1991: Klimisch 2), which reported increased frequencies of micronuclei at mM concentrations, and an in vivo mouse bone marrow micronucleus assay, which at i.p. doses up to the maximum tolerated dose did not show any potential for clastogenicity (1990: Klimisch 1). The in vivo assay is more relevant for human health hazard assessment, so that it is concluded that IPHA does not exert any relevant potential for clastogenicity. The results on in vitro gene mutation by IPHA were partly equivocal, however, the overall weight of evidence from all genotoxicity studies indicates that purity and handling of the test material is critical to evaluate the genotoxicity potential of IPHA. Due to IPHA’s potential to decompose, some of the studies most probably detected genotoxic impurities/decomposition products.Hence, following a re-evaluation of the available evidence, it is concluded that it is unlikely that IPHA possesses a relevant potential for mutagenicity.
Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 - 23 January 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to test guidelines and in accordance with GLP
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
METI Act No. 117, Ministerial Ordinance No. 1-3 (2004)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Salmonella typhimurium histidine auxotrophs and and Escherichia coli WP2 uvrA
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system.
Test concentrations with justification for top dose:
1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 ug/plate for all strains with and without S9 in introductory assay.
50,150,500,1500 and 5000 ug/plate for all strains with and without S9 in confirmatory assay.
Vehicle / solvent:
water
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-nitrofluorene (TA98), sodium azide (TA100 and TA1535), 9-Aminoacridine (TA1537) and methyl methanesulfona (WP2uvrA) without S9 and 2-aminoanthracene (all strains) with S9
Details on test system and experimental conditions:
Initial Toxicity-Mutation Assay
The initial toxicity-mutation assay was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. Vehicle control, positive controls and a minimum of eight dose levels of the test article were plated, two plates per dose, with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA on selective minimal agar in the presence and absence of Aroclor-induced rat liver S9.

Confirmatory Mutagenicity Assay
The confirmatory mutagenicity assay was used to evaluate and confirm the mutagenic potential of the test article. A minimum of five dose levels of test article along with appropriate vehicle control and positive controls were plated with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA on selective minimal agar in the presence and absence of Aroclor-induced rat liver S9. All dose levels of test article, vehicle control and positive controls were plated in triplicate.

Plating and Scoring Procedures
The test system was exposed to the test article via the preincubation methodology described by Yahagi et al. (1977). On the day of its use, minimal top agar, containing 0.8 % agar (W/V) and 0.5 % NaCl (W/V), was melted and supplemented with L-histidine, D-biotin and L-tryptophan solution to a final concentration of 50 μM each. Top agar not used with S9 or Sham mix was supplemented with 25 mL of water for each 100 mL of minimal top agar. For the preparation of media and reagents, all references to water imply deionized water. Bottom agar was Vogel-Bonner minimal medium E (Vogel and Bonner, 1956) containing 1.5 % (W/V) agar. Nutrient bottom agar was Vogel-Bonner minimal medium E containing 1.5 % (W/V) agar and supplemented with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder). Nutrient Broth was Vogel-Bonner salt solution supplemented with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder).

Each plate was labeled with a code system that identified the test article, test phase, concentration, tester strain and activation, as described in detail in BioReliance's Standard Operating Procedures.

One-half (0.5) milliliter of S9 or sham mix, 100 μL of tester strain (cells seeded) and 100 μL of vehicle or test article dilution were added to 13 X 100 mm glass culture tubes pre-heated to 37±2°C. After vortexing, these mixtures were incubated with shaking for 20±2 minutes at 37±2°C. Following the preincubation, 2.0 mL of selective top agar was added to each tube and the mixture was vortexed and overlaid onto the surface of 25 mL of minimal bottom agar. When plating the positive controls, the test article aliquot was replaced by a 50 μL aliquot of appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for approximately 48 to 72 hours at 37±2°C. Plates that were not counted immediately following the incubation period were stored at 2-8°C until colony counting could be conducted.

Evaluation criteria:
The condition of the bacterial background lawn was evaluated for evidence of test article toxicity by using a dissecting microscope. Precipitate was evaluated by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate using the codes shown in the following table.

Revertant colonies for a given tester strain and activation condition, except for positive controls, were counted either entirely by automated colony counter or entirely by hand unless the plate exhibited toxicity.

For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.

For the test article to be evaluated positive, it must cause a reproducible, concentration-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article. Data sets for tester strains TA98, TA1535, TA1537 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the response was greater than or equal to 3.0-times the mean vehicle control value. Data sets for tester strain TA100 were judged positive if the increase in mean revertants at the peak of the response was greater than or equal to 2.0-times the mean vehicle control value.

An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative, if it was neither positive nor equivocal.

Statistics:
Statistical analysis was not conducted.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Solubility Test
Water was selected as the solvent of choice based on the Sponsor’s request, solubility of the test article and compatibility with the target cells. The test article formed a clear solution in water at approximately 50 mg/mL, the maximum concentration tested in the solubility test.

Sterility Results
No contaminant colonies were observed on the sterility plates for the vehicle control, the test article dilutions and the S9 and Sham mixes.

Initial Toxicity-Mutation Assay
In Experiment B1 (Initial Toxicity-Mutation Assay), the maximum concentration tested was 5000 μg per plate, which is the maximum concentration recommended by test guidelines. This concentration was achieved using a concentration of 50 mg/mL and a plating aliquot of 100 μL. The concentrations tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Neither precipitate nor background toxicity was observed. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 μg per plate.

Confirmatory Mutagenicity Assay
In Experiment B2 (Confirmatory Mutagenicity Assay), no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. The dose levels tested were 50, 150, 500, 1500 and 5000 μg per plate. Neither precipitate nor background lawn toxicity was observed.

Dosing Formulation Analysis
Dosing formulations were analyzed by the Sponsor. Concentration analysis indicates that the actual mean concentrations of the analyzed dose levels were between 95.7 and 109.5% of their respective targets with a relative percent difference (RPD) of < 20%. This indicates that the regulatory-required top dose level was achieved and the results support the validity of the study conclusion. No test article was detected in the vehicle control sample.

Stability analysis was performed on the highest and lowest dose concentrations in the initial toxicity-mutation assay, and the results of the analysis for the lowest dose concentration were outside the acceptable range of 85 to 115% of dose (start). The stability analysis was repeated with samples from the confirmatory mutagenicity assay at concentrations ranging from 0.015 to 50 mg/mL. The results of the analysis on the formulations from the confirmatory assay found the test article to be stable in water at room temperature for the period of dosing.

Table 1

Initial Toxicity-Mutation Assay without S9 activation        

     Mean revertants +St. Dev./plate            
  Article   Dose level per plate  TA98  TA100  TA1535  TA1537

WP2 uvrA

 IPHA

 5000 ug  16 + 1  126 + 6  10 + 2  6 + 3  34 + 0
   1500 ug  16 + 5  118 + 0  8 + 1  3 + 2  33 + 0
   500 ug  16 + 5  92 + 19  17 + 3  3 + 3  24 + 1
   150 ug  17 + 3  106 + 6  8 + 1  7 + 3  30 + 4
   50 ug  14 + 4  99 + 8  8 + 6  3 + 3  35 + 2
   15 ug  12 + 6  116 + 13  7 + 2  4 + 1  32 + 1
   5.0 ug  14 + 4  89 + 6  8 + 1  6 + 4  35 + 4
   1.5 ug  14 + 3  108 + 10  8 + 1  3 + 0  32 + 7
 Water  100 uL  12 + 1  93 + 1  10 + 2  5 + 3  44 + 3
 2 -Nitrofluorene  1.0 ug  549 + 34  ND  ND  ND  ND
 Sodium Azide  1.0 ug  ND  684 + 69  417 + 3  ND  ND
 9 -Aminoacridine  75 ug  ND  ND  ND

 542 + 20

 ND
 Methyl Methanesulfonate  1000 ug  ND  ND  ND  ND

 392 + 65

ND = No Data

Table 2

Initial Toxicity-Mutation Assay with S9 activation

     Mean revertants +St. Dev./plate            
  Article   Dose level per plate  TA98  TA100  TA1535  TA1537

WP2 uvrA

 IPHA

 5000 ug  17 + 6  124 + 4  8 + 0  5 + 5  52 + 9
   1500 ug  15 + 4  129 + 13  13 + 1  8 + 1  34 + 4
   500 ug  10 + 1  110 + 11  15 + 2  4 + 0  26 + 12
   150 ug  12 + 4  106 + NA  9 + 3  2 + 1  35 + 2
   50 ug  13 + 3  101 + 4  11 + 8  1 + 0  29 + 6
   15 ug  18 + 8  106 + 12  12 + 1  5+ 3  35 + 4
   5.0 ug  8 + 1  128 + 8  11 + 2  8+ 5  37 + 1
   1.5 ug  11 + 6  100 + 14  9 + 5  4+ 1  34 + 6
 Water  100 uL  12 + 6  106 + 28  8 + 1  6 + 2  31 + 3
 2 -Aminoanthracene  1.0 ug  129 + 6  ND  55 + 4  41 + 6  ND
 2 -Aminoanthracene  2.0 ug  ND  446 + 62  ND  ND  ND
  2 -Aminoanthracene  15 ug  ND  ND  ND

 ND

 455 + 34

ND = No Data

Table 3

Confirmatory Mutagenicity Assay without S9 activation

     Mean revertants +St. Dev./plate            
  Article   Dose level per plate  TA98  TA100  TA1535  TA1537

WP2 uvrA

 IPHA

 5000 ug  14 + 5  141 + 18  13 + 2  4 + 2  19 + 7
   1500 ug  16 + 6  111 + 8  9 + 2  5 + 4  17 + 7
   500 ug  10 + 2  107 + 12  11 + 1  4 + 3  22 + 7
   150 ug  12 + 5  110 + 8  12 + 4  4 + 2  14 + 3
   50 ug  9 + 3  106 + 3   11 + 1  6 + 3  16 + 6
 Water  100 uL  12 + 2  125 + 13  15 + 4   5 + 1  16 + 5
 2 -Nitrofluorene  1.0 ug  451 + 114  ND  ND  ND  ND
 Sodium Azide  1.0 ug  ND  650 + 31  530 + 27  ND  ND
 9 -Aminoacridine  75 ug  ND  ND  ND

 305 + 70

 ND
 Methyl Methanesulfonate  1000 ug  ND  ND  ND  ND

 299 + 32

ND = No Data

Table 4

Confirmatory Mutagenicity Assay with S9 activation

     Mean revertants +St. Dev./plate            
  Article   Dose level per plate  TA98  TA100  TA1535  TA1537

WP2 uvrA

 IPHA

 5000 ug  15 + 6  151 + 6  6 + 2  8 + 4  23 + 5
   1500 ug  15 + 6  120 + 10  7 + 2  6 + 3  17 + 4
   500 ug  13 + 6  138 + 9  12 + 1  5 + 1  24 + 8
   150 ug  14 + 3  117 + 8  6 + 3  9 + 1  25 + 3
   50 ug  8 + 2  138 + 25   11 + 3  4 + 1  18 + 2
 Water  100 uL  10 + 9  149 + 22  13 + 4   7 + 5  20 + 4
 2 -Aminoanthracene  1.0 ug  540 + 85  ND  58 + 18  80 + 6  ND
 2- Aminoanthracene  2.0 ug  ND  857 + 79  ND  ND  ND
 2 -Aminoanthracene  15 ug  ND  ND  ND

 ND

 98 + 5

ND = No Data

Conclusions:
All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, IPHA (Isopropylhydroxylamine) was negative (non-mutagenic) both in the presence and absence of
Aroclor-induced rat liver S9.
Executive summary:

The test article, IPHA (Isopropylhydroxylamine), was tested in the Bacterial Reverse Mutation Assay using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA in the presence and absence of Aroclor-induced rat liver S9. The assay was performed in two phases using the preincubation method. The first phase, the initial

toxicity-mutation assay, was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test article.

Water was selected as the solvent of choice based on the Sponsor’s request, solubility of the test article and compatibility with the target cells. The test article formed a clear solution in water at approximately 50 mg/mL, the maximum concentration tested in the solubility test.

In the initial toxicity-mutation assay, the maximum concentration tested was 5000 μg per plate, which is the maximum concentration recommended by test guidelines. This concentration was achieved using a concentration of 50 mg/mL and a plating aliquot of 100 μL. The concentrations tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate. No positive mutagenic responses

were observed with any of the tester strains in either the presence or absence of S9 activation. Neither precipitate nor background lawn toxicity was observed. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 μg per plate.

In the confirmatory mutagenicity assay, no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. The dose levels tested were 50, 150, 500, 1500 and 5000 μg per plate. Neither precipitate nor background lawn toxicity was observed.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

There have been seven reports/publications reporting 10 assays conducted to evaluate the genotoxic potential of IPHA. Four of these 10 studies were bacterial gene-mutation assays, four were gene mutation/DNA repair indicator assays in mammalian cells, one study an in vitro clastogenicity study. The 10th study was an in vivo micronucleus study in mice.

Concerning the bacterial gene mutation endpoint, the key study Ames test was negative. This study was conducted according to current test guidelines and GLP with 99.7% pure IPHA. Two of the supporting Ames studies (1986: Klimisch 2 and 1985: Klimisch 4) were negative as well. One study performed with a 15% watery solution of IPHA (Klimisch 4) reported mutagenicity. As three other studies were negative, and it is probable that the findings of the study were due to decomposition products formed in watery solutions of IPHA upon improper storage, it is concluded that IPHA does not possess relevant potential to cause bacterial gene mutations.

For mammalian cell gene mutation, one HGPRT assay was reported negative (1985: Klimisch 4), another HGPRT assay indicated a positive response (Haas-Jobelius et al. 1991: Klimisch 2), and the most recent CHO/HGPRT (2014: Klimisch 1) was negative. An in vitro rat hepatocyte UDS DNA repair assay also conducted by Haas-Jobelius 1991 was negative. 

The clastogenicity endpoint was addressed by an in vitro V79 micronucleus assay by Haas-Jobelius et al. (1991: Klimisch 2), which reported increased frequencies of micronuclei at mM concentrations, and an in vivo mouse bone marrow micronucleus assay, which at i.p. doses up to the maximum tolerated dose did not show any potential for clastogenicity (1990: Klimisch 1). The in vivo assay is more relevant for human health hazard assessment, so that it is concluded that IPHA does not exert any relevant potential for clastogenicity.

The results on mammalian cell in vitro gene mutation by IPHA were equivocal, however, the overall weight of evidence from all genotoxicity studies indicates that purity and handling of the test material is critical to evaluate the genotoxicity potential of IPHA. Due to IPHA’s potential to decompose, some of the studies most probably detected genotoxic impurities/decomposition products.The test material used by Haas-Jobelius was 97% pure, with storage conditions of the test material and stock/test solutions not reported. The 2014 OECD guideline/GLP compliant HGPRT with 99% IPHA did not detect any gene mutations, so that the weight of evidence on mammal cell line gene mutation studies is also negative.

Currently, there is no data existing on the metabolism of IPHA in rat or any other species. On the basis of the chemical structure, IPHA contains the two moieties of isopropyl and hydroxylamine and its metabolism has been predicted based on the metabolism of both isopropyl and hydroxylamine. The isopropyl group can be metabolized to by cytochrome P450 via hydroxylation, this will lead to the hydroxylated IPHA metabolite. The hydroxylamine group is expected to be metabolically stable, and will not be further metabolized by cytochrome P450 or other Phase I or II enzymes,in vivo or in vitro. Based on these rationales, the potential metabolite of IPHA will be hydroxylated isopropylhydroxyamine.It is therefore unlikely that IPHA metabolites are responsible for the sporadic positive findings in genotoxicity assays, which is also supported by the fact that such findings occurred both in the presence and absence of external metabolic activation systems.

Hence, following a re-evaluation of the available evidence, it is concluded that it is unlikely that IPHA possesses a relevant potential for mutagenicity.


Justification for selection of genetic toxicity endpoint
The study was conducted according to test guidelines and in accordance with GLP

Justification for classification or non-classification

IPHA is unlikely to exert any relevant potential of genotoxicity and one in vivo genotoxicity assay was negative so that classification for the muatgenicity endpoint is not warranted.

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